The non-receptor protein tyrosine kinases p56lck and p59fyn are pivotal regulators of normal signaling processes in lymphocytes. Previous studies have demonstrated that p56lck interacts physically and functionally with numerous T cell receptor structures, including the CD4 and CD8 coreceptors, and the IL-2 receptor beta chain. More recent experiments have defined an exquisitely specific function for this kinase: p56lck directs the development of immature T-lineage cells by sensing the products of functional rearrangement of T cell receptor beta chain genes, and stimulating thymocyte proliferation (and maturation) thereafter. Indeed, interdiction of the lck signaling pathway both blocks thymocyte differentiation and compromises allelic exclusion at the T cell receptor beta loci. This proposal seeks continued support for the elucidation of signaling pathways to which p56lck, and the closely related kinase p59fyn, contribute. First, a series of mouse mutants will be evaluated to learn whether p56lck acts primarily by phosphorylating T cell receptor components. Efforts will then be made to establish a signaling system through which p56lck kinase activity can be experimentally manipulated. The consequences of these manipulations will be investigated in thymocytes, and a parallel analysis will be undertaken in cell lines. A system will also be devised that will permit interference with p56lck function in mature T lymphocytes. Lastly, the function of p59fyn will be addressed through reconstitution of existing mouse mutants. The (very) long-range goal of these studies is the complete dissection of signaling pathways that regulate lymphocyte activation, pathways wherein these two non-receptor protein tyrosine kinases plan crucial roles.